Synthesis, Antifungal Ergosterol Inhibition, Antibiofilm Activities, and Molecular Docking on ß-Tubulin and Sterol 14-Alpha Demethylase along with DFT-Based Quantum Mechanical Calculation of Pyrazole Containing Fused Pyridine-Pyrimidine Derivatives.

Multidrug-resistant fungal infections have become much more common in recent years, especially in immune-compromised patients. Therefore, researchers and pharmaceutical professionals have focused on the development of novel antifungal agents that can tackle the problem of resistance. In continuation to this, a novel series of pyrazole-bearing pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione derivatives (4a-4o) have been developed. These compounds have been screened against Candida albicans, Aspergillus niger, and Aspergillus clavatus. The synthesized compounds were characterized by well-known spectroscopic techniques, i.e., IR, 1H NMR, 13C NMR, and mass spectrometry. In vitro antifungal results revealed that compound 4n showed activity against C. albicans having MIC value of 200 µg/mL. To know the plausible mode of action, the active derivatives were screened for anti-biofilm and ergosterol biosynthesis inhibition activities. The compounds 4h, 4j, 4k, and 4n showed greater ergosterol biosynthesis inhibition than the control DMSO. To comprehend how molecules interact with the receptor, studies of molecular docking of 4k and 4n have been performed on the homology-modeled protein of ß-tubulin. The molecular docking revealed that the active compounds 4h, 4j, 4k, 4l, and 4n interacting with the active site amino acid of sterol 14-alpha demethylase (PDB ID: 5v5z) indicate one of the possible modes of action of ergosterol inhibition activity. The synthesized compounds 4c, 4e, 4h, 4i, 4j, 4k, 4l, and 4n inhibited biofilm formation and possessed the potential for anti-biofilm activity. DFT-based quantum mechanical calculations were carried out to optimize, predict, and compare the vibration modes of the molecule 4a.

Design, synthesis, antimicrobial activity, DFT, and molecular docking studies of pyridine-pyrazole-based dihydro-1,3,4-oxadiazoles against various bacterial and fungal targets.

Antimicrobial resistance which is increasing at an alarming rate is a severe public health issue worldwide. Hence, the development of novel antibiotics is an urgent need as microbes have developed resistance against available antibiotics. In search of novel antimicrobial agents, a convenient route for the preparation of substituted 3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-(2-phenyl-5-(pyridin-3-yl)-1,3,4-oxadiazol-3(2H)-yl)prop-2-en-1-ones (6a-6o) has been adopted by using pyridine-3-carbohydrazide and various aromatic aldehydes. The newly synthesized compounds were characterized by using various spectral techniques, for example, IR, 1 H NMR, 13 C NMR, and mass spectroscopy. Synthesized hybrids were studied for in vitro antimicrobial potency against various bacterial and fungal strains. Antibacterial results revealed that compounds 6e, 6h, 6i, 6l, and 6m were found to be most active against bacterial strains as they showed minimum inhibitory concentration (MIC) value of 62.5 µg/mL while compounds 6d, 6e, and 6h showed MIC value of 200 µg/mL against Candida albicans. The quantum parameters that relate to the bioavailability of the compounds were computed, followed by docking with different bacterial and fungal targets like sortase A, dihydrofolate reductase, thymidylate kinase, gyrase B, sterol 14-alpha demethylase. The experimental and computational results are in good agreement.

Characterization, Anti-proliferative Activity, and Bench-Scale Production of Novel pH-Stable and Thermotolerant L-Asparaginase from Bacillus licheniformis PPD37.

Bacterial L-asparaginase (LA) is a chemotherapeutic drug that has remained mainstay of cancer treatment for several decades. LA has been extensively used worldwide for the treatment of acute lymphoblastic leukemia (ALL). A halotolerant bacterial strain Bacillus licheniformis sp. isolated from marine environment was used for LA production. The enzyme produced was subjected to purification and physico-chemical characterisation. Purified LA was thermotolerant and demonstrated more than 90% enzyme activity after 1 h of incubation at 80 °C. LA has also proved to be resistant against pH gradient and retained activity at pH ranging from 3.0 to 10. The enzyme also had high salinity tolerance with 90% LA activity at 10% NaCl concentration. Detergents like Triton X-100 and Tween-80 were observed to inhibit LA activity while more than 70% catalytic activity was maintained in the presence of metals. Electrophoretic analysis revealed that LA is a heterodimer (~ 63 and ~ 65 kDa) and has molecular mass of around 130 kDa in native form. The kinetic parameters of LA were tested with LA having low Km value of 1.518 µM and Vmax value of 6.94 µM/min/mL. Purified LA has also exhibited noteworthy antiproliferative activity against cancer cell lines-HeLa, SiHa, A549, and SH-SY-5Y. In addition, bench-scale LA production was conducted in a 5-L bioreactor using moringa leaves as cost-effective substrate.

Benzothiazole Clubbed Imidazolone Derivatives: Synthesis, Molecular Docking, DFT Studies, and Antimicrobial Studies.

AIM: This study aims to synthesize antimicrobial agents and their molecular docking, and DFT studies of benzothiazole-imidazolone scaffolds. BACKGROUND: Benzothiazole and imidazolone analogues are of interest due to their potential activity against microbial infections. In search of suitable antimicrobial compounds, we report here the synthesis, characterization, and biological activities of benzothiazole and imidazolone analogues (4a-l). OBJECTIVE: The benzothiazole clubbed imidazolone motifs were synthesized, characterized, and screened for their antimicrobial activity. Molecular docking was carried out for the development of antimicrobial agents based on the results of biological activity obtained. METHODS: We have synthesized a new series of benzothiazole-clubbed imidazolone hybrids by using multi-step reactions in the search for antimicrobial agents (4a-l). The structures were determined by 1H NMR, 13C NMR, IR, and mass spectroscopy techniques. Moreover, synthesized compounds were evaluated for their antimicrobial activity by using a Serial Broth Dilution method. In addition, molecular electrostatic potential, geometric optimization, and molecular reactivity analyses (HOMO-LUMO) of 4c, which is one of the compounds with the highest antibacterial activity, were performed. RESULTS: The in vitro antimicrobial activity was evaluated against pathogenic strains. Among them, compounds 4c showed the most potent biological activity against Gram-negative bacteria, E. coli with MIC values of 50 µg/mL, and compound 4c active against A. clavatus with MIC values of 100 µg/mL. Active compound 4c HUMO-LUMO energies, molecular electrostatic potential analysis, and geometric optimization parameters were calculated with a 6-31G ** base set using DFT/B3LYP theory, and the results were displayed. Molecular docking studies were performed on E. coli DNA Gyrase B to understand the binding interaction of compound 4c, and it was observed that compound 4c interacted with Arg76 amino acid of the active site through hydrophobic interaction. CONCLUSION: Benzothiazole-clubbed imidazolone hybrids (4a-l) indicated promising antimicrobial activity. Among them, compounds 4b (MIC=50 µg/mL C. albicans), 4c (MIC=50 µg/mL, E. coli), 4e (MIC= 100 µg/mL, A. niger), and 4g (MIC= 50 µg/mL, S. pyogenes) with electronwithdrawing bromo, chloro, and fluoro group at the para position of the phenyl ring on benzothiazole-imidazolone hybrids indicated remarkable potency compared to the standard drug. The geometric optimization, molecular reactivity, and MESP analyses of 4c were calculated with the B3LYP/6-31G ** base set and ΔE = ELUMO-EHOMO, which was found to be - 0.12096 eV. In addition, the binding affinity scores correlated well with the in vitro antimicrobial activity (4c), while their binding modes proposed the involvement of steric, electrostatic, and hydrogen- bonding interactions with the active site.

Assessment of surface water quality during different tides and an anthropogenic impact on coastal water at Gulf of Kachchh, West Coast of India.

The port-based activity is often associated with industrial growth in the hinterland and similar phenomenon reported from the Gulf of Kachchh, India. Industrialization exerts pressure on coastal water through the release of waste water or effluents which influence the entire marine ecosystem. The present paper tries to evaluate the variation in the water quality during the high tide and low tide in relation to the anthropogenic or natural influence in Gulf of Kachchh. The tidal variation is important as it reflects the influence of the land-based activity on the coastal waters. To prove this logic, a series of stations were taken along the coastal water and statistical analysis, viz., Pearson correlation, Box plot, hierarchical cluster analysis (HCA), and factor analysis (PCA/FA) were conducted. Pearson correlation and Box plot represent visual impact of parameter variations in respected tides. The chemometric analysis, i.e., HCA and PCA/FA, clearly indicates an anthropogenic impact on coastal water. The results of HCA revealed that major anthropogenic and domestic impacts were found at various stations during the low tide. The HCA points out that an anthropogenic and the tidal activity in the Gulf of Kachchh influence the physical water quality parameters like pH, salinity, dissolved solid, oxygen, turbidity, sulfate, and nutrients in the coastal ecosystem. The PCA/FA further ascertains the finding of HCA analysis that the state of the art of the water quality of coastal ecosystem has direct relevance with the land-based activities and sewage outfall points. Tide-based control on the water quality parameters was evident that the high tide nutrients like phosphates and nitrogen were high, while during the low tide, temperature, salinity, total solids, and sulfate showed higher concentrations. The findings of the paper will be useful for developing effective management strategies for policy makers or stakeholders operating in the coastal area.

Exploring bacterial communities through metagenomics during bioremediation of polycyclic aromatic hydrocarbons from contaminated sediments.

The goal of this study was to evaluate the degradation effectiveness of PAHs degrading bacteria at the mesocosm level, including Stenotrophomonas maltophilia (SC), mixed culture (MC), and enriched native microflora (EC) at the mesocosm level. Maximum degradation was found in the mesocosm MC (26.67 %), followed by SC (25.08 %) and EC (18.25 %) after 60 days. Thus, mixed culture and Stenotrophomonas maltophilia could be a game changer in the PAHs bioremediation at the chronically contaminated sites. MiSeq sequencing has revealed dominancy of γ-Proteobacteria, α-Proteobacteria, ß-Proteobacteria at class level and Sphingomonadales, oceanospirillales, Rhodothermales at Order level. Families Alcanivoracaceae, Alteromonadaceae, Nocardiaceae, Rhodospirillaceae and genus Stenotrophomonas, Alcanivorax, Methylophaga, Fluviicola and Rhodoplanes were considerably increased which play key role in the PAHs degradation. Dominant bacterial communities have revealed resilience community to enable potential PAHs degradation process in all the mesocosms. To the best our knowledge this is the first ever attempt in PAHs biodegradation study conducted at the mesocosm level mimicking natural environmental conditions. Consequently, this study could be a benchmark against which future progress studies for the policy makers and stakeholders to design appropriate bioremediation study for the historically PAHs polluted contaminate sites.

Development of Process and Data Centric Inference System for Enhanced Production of L-Asparaginase from Halotolerant Bacillus licheniformis PPD37.

The present study aims at bioengineering of medium components using data and process centric approaches for enhanced production of L-asparaginase, an important biological molecule, by halotolerant Bacillus licheniformis PPD37 strain. To achieve this, first significant medium components were screened followed by optimisation of a combination of media components and culture conditions such as L-asparagine, MgSO4, NaCl, pH, and temperature. Optimisation study was carried out using statistical models such as response surface methodology (RSM) - process centric and artificial neural network (ANN) - data centric approaches. The production improved from 2.86 U/mL to 17.089 U/mL, an increase of approximately 6-times of the unoptimised L-asparaginase production. On comparing RSM and ANN models for optimised L-asparaginase production based on R2 value, mean absolute percentage error (MAPE), root mean square error (RMSE), and mean absolute deviation (MAD) values, the ANN model emerged as the superior one. As this is the first report to the authors best knowledge on development of inference system using RSM and ANN models for enhanced L-asparaginase production using a halotolerant bacteria, this study could lead to more in-depth and large-scale L-asparaginase production.

Assessment of surface water of Gulf of Kachchh, west coast of India: A chemometric approach.

The present study is aimed at investigation of surface water quality of Gulf of Kachchh (GoK), Gujarat. The main objective of this study was to convert complex dataset of water quality parameters from GoK into comprehensive, simple and interpretable observations. Hence, physico-chemical parameters and nutrients of surface water from GoK were analyzed. Chemometric results indicated that oxygen, salinity, dissolved solids, nutrient and natural conditions were the factors that affected surface water quality. The water quality index was calculated to identify water quality classes to evaluate the spatio-seasonal trend in the study area. The results revealed that water quality was moderate in summer, worst in pre-monsoon and best in post-monsoon. The study also highlighted that Marine National Park (Central GoK) was observed to be comparatively in good condition with abundant marine biodiversity. Thus, the results of chemometric study of water quality parameters can be a valuable tool for government authorities for sustainable development of GoK.

Antibiotic resistance genes allied to the pelagic sediment microbiome in the Gulf of Khambhat and Arabian Sea.

Antibiotics have been widely spread in the environments, imposing profound stress on the resistome of the residing microbes. Marine microbiomes are well established large reservoirs of novel antibiotics and corresponding resistance genes. The Gulf of Khambhat is known for its extreme tides and complex sedimentation process. We performed high throughput sequencing and applied bioinformatics techniques on pelagic sediment microbiome across four coordinates of the Gulf of Khambhat to assess the marine resistome, its corresponding bacterial community and compared with the open Arabian Sea sample. We identified a total of 2354 unique types of resistance genes, with most abundant and diverse gene profile in the area that had anthropogenic activities being carried out on-shore. The genes with >1% abundance in all samples included carA, macB, sav1866, tlrC, srmB, taeA, tetA, oleC and bcrA which belonged to the macrolides, glycopeptides and peptide drug classes. ARG enriched phyla distribution was quite varying between all the sites, with Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes among the dominant phyla. Based on the outcomes, we also propose potential biomarker candidates Desulfovibrio, Thermotaga and Pelobacter for antibiotic monitoring in the two of the Gulf samples probable contamination prone environments, and genera Nitrosocccus, Marinobacter and Streptomyces in the rest of the three studied samples. Outcomes support the concept that ARGs naturally originate in environments and human activities contribute to the dissemination of antibiotic resistance.

Concentrations, input prediction and probabilistic biological risk assessment of polycyclic aromatic hydrocarbons (PAHs) along Gujarat coastline.

A comprehensive investigation was conducted in order to assess the levels of PAHs, their input prediction and potential risks to bacterial abundance and human health along Gujarat coastline. A total of 40 sediment samples were collected at quarterly intervals within a year from two contaminated sites-Alang-Sosiya Shipbreaking Yard (ASSBRY) and Navlakhi Port (NAV), situated at Gulf of Khambhat and Gulf of Kutch, respectively. The concentration of ΣPAHs ranged from 408.00 to 54240.45 ng g-1 dw, indicating heavy pollution of PAHs at both the contaminated sites. Furthermore, isomeric ratios and principal component analysis have revealed that inputs of PAHs at both contaminated sites were mixed-pyrogenic and petrogenic. Pearson co-relation test and regression analysis have disclosed Nap, Acel and Phe as major predictors for bacterial abundance at both contaminated sites. Significantly, cancer risk assessment of the PAHs has been exercised based on incremental lifetime cancer risks. Overall, index of cancer risk of PAHs for ASSBRY and NAV ranged from 4.11 × 10-6-2.11 × 10-5 and 9.08 × 10-6-4.50 × 10-3 indicating higher cancer risk at NAV compared to ASSBRY. The present findings provide baseline information that may help in developing advanced bioremediation and bioleaching strategies to minimize biological risk.

Characterization of L-asparaginase from marine-derived Aspergillus niger AKV-MKBU, its antiproliferative activity and bench scale production using industrial waste.

L-asparaginase (LA), an enzyme with anticancer activities, produced by marine-derived Aspergillus niger was subjected to purification and characterization. The purified enzyme was observed to have molecular weight ∼90KDa. The enzyme retained activity over a wide range of pH, i.e. pH 4-10. The enzyme was quite stable in temperature range 20-40°C. Tween 80 and Triton X-100 were observed to enhance LA activity while inhibition of LA activity was observed in presence of heavy metals. The values for Km was found to be 0.8141 mM and Vmax was 6.228µM/mg/min. The enzyme exhibited noteworthy antiproliferative activity against various cancer cell lines tested. Successful bench scale production (in 5L bioreacator) of LA using groundnut oil cake as low cost substrate has also been carried out.

Description of Rhodobacter azollae sp. nov. and Rhodobacter lacus sp. nov.

Three strains (JA826T, JA912T and JA913), which were yellowish brown colour, rod to oval shaped, Gram-stain-negative, motile, phototrophic bacteria with a vesicular architecture of intracytoplasmic membranes, were isolated from different pond samples. The DNA G+C content of the three strains was between 64.6 and 65.5 mol%. The highest 16S rRNA gene sequence similarity of all three strains was with the type strains of the genus Rhodobacter sensu stricto in the family Rhodobacteraceae. Strain JA826T had highest sequence similarity with Rhodobacter maris JA276T (98.5 %), Rhodobacter viridis JA737T (97.5 %) and other members of the genus Rhodobacter (<97 %). Strain JA912T had highest sequence similarity with Rhodobacter viridis JA737T (99.6 %), Rhodobacter sediminis N1T (99.3 %), Rhodobacter capsulatus ATCC 11166T (98.8 %) and less than 97 % similarity with other members of the genus Rhodobacter. The 16S rRNA gene sequence similarity between strains JA826T and JA912T was 96.9 %. DNA-DNA hybridization showed that strains JA826T and JA912T (values among themselves and between the type strains of nearest members <44 %) did not belong to any of the nearest species of the genus Rhodobacter. However, strains JA912T and JA913 were closely related (DNA-DNA hybridization value >90 %). The genomic distinction was also supported by differences in phenotypic and chemotaxonomic characteristics in order to propose strains JA826T (=KCTC 15478T=LMG 28758T) and JA912T (=KCTC 15475T=LMG 28748T) as new species in the genus Rhodobacter sensu stricto with the names Rhodobacter lacus and Rhodobacter azollae, respectively.

Description of a phototrophic bacterium, Thiorhodococcus alkaliphilus sp. nov.

Strain JA878T was purified from a photoheterotrophic enrichment obtained from a sediment sample of a brown pond near Nari Salt Pan, Bhavnagar, Gujarat, India. Cells of the isolate were coccoid, motile by means of single polar flagellum and Gram-stain-negative. The internal photosynthetic membrane architecture was vesicular. Strain JA878T contained bacteriochlorophyll a and spirilloxanthin series of carotenoids with rhodopin (>85 %) as the major component. Strain JA878T grew optimally at pH 10-11, and had no requirement for NaCl (tolerated up to 6 %, w/v) or vitamins for growth. C16 : 1ω7c/C16 : 1ω6c, C18 : 1ω7c/C18 : 1ω6c and C16 : 0 were identified as the major fatty acids (>10 %). Phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and an unknown polar lipid were identified. Q8 was the predominant quinone system in strain JA878T. The DNA G+C content was 62.4 mol%. Highest 16S rRNA gene sequence similarity through EzTaxon-based blast analysis of strain JA878T was found with the type strains of Thiorhodococcus fuscus (99 %), Thiorhodococcus kakinadensis (98.6 %), Thiohalobacter thiocyanaticus (98.4 %), Thiophaeococcus fuscus (97.3 %) and other members of the class Gammaproteobacteria (<97.3 %), revealing a close affiliation to the genera Thiorhodococcus, Thiohalobacter and Thiophaeococcus. However, the phylogenetic treeing firmly placed the strain in the genus Thiorhodococcus. Phenotypic and chemotaxonomic evidence supported the affiliation of strain JA878T to the genus Thiorhodococcus and not to Thiohalobacter, Thiophaeococcus or other known genera of Chromatiaceae. Distinct physiological, genotypic and chemotaxonomic differences indicate that strain JA878T represents a novel species of the genus Thiorhodococcus, for which the name Thiorhodococcus alkaliphilus sp. nov. is proposed. The type strain is JA878T (=KCTC 15531T=JCM 31245T).

Polycyclic aromatic hydrocarbons (PAHs) at the Gulf of Kutch, Gujarat, India: Occurrence, source apportionment, and toxicity of PAHs as an emerging issue.

The present study extrapolates the assessment and characterization of a barely studied region, the Gulf of Kutch, (near Jamnagar), Gujarat, India, in terms of PAH exposure, adverse effects caused by them, and various toxicological indices showing the catastrophic effects of their elevated concentrations. ΣPAH concentration in the site ranged from 118,280 to 1,099,410ngg-1 dw, with a predominance of 2-3-ring PAHs (79.09%) as compared to 4-5- and 6-ring PAHs (20.91%). The concentrations of carcinogenic PAHs were found to be between 8120 and 160,000ngg-1 dw, with a mean of 63,810ngg-1 dw, which is much higher than normal acceptable values. The toxic equivalent quotient for 6CPAHs ranged from 150.47 to 26,330ngg-1 BaPeq, encompassing 50.63% of ΣPAH toxicity. This toxicological profile of the present study site would be of paramount importance as it offers fresh information regarding the load of legacy pollutants such as PAHs and the inputs and methods to cope with their extremely high concentrations in less explored marine habitats.

Rhodovulum algae sp. nov., isolated from an algal mat.

A reddish-brown-pigmented, phototrophic bacterium, designated strain JA877T, was isolated from a brown algae mat sample collected from Jalandhar beach, Gujarat, India. On the basis of the 16S rRNA gene sequence, strain JA877T belongs to the class Alphaproteobacteria and is closely related to the type strains Rhodovulum viride JA756T (99.0 %), Rhodovulum sulfidophilum Hansen W4T (98.9 %), Rhodovulumvisakhapatnamense JA181T (98.8 %),Rhodovulum kholense JA297T (97.5 %) and Rhodovulum salis JA746T (97.0). However, strain JA877T showed only 20-45 % relatedness with its phylogenetic neighbours and had a ∆Tm between 5.8 and 7.0 °C. The major respiratory quinone was ubiquinone-10 (Q10), and the polar lipid profile was composed of the major components phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, two unidentified sulfolipids and five unidentified lipids. The major fatty acids were C18 : 1ω5c, C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C18 : 0. The DNA G+C content was 64.5 mol%. On the basis of 16S rRNA gene sequence analysis, physiological data, and chemotaxonomic and molecular differences, strain JA877T is significantly different from other species of the genus Rhodovulum and represents a novel species, for which the name Rhodovulum algae sp. nov. is proposed. The type strain is JA877T (=LMG 29228T= KCTC 42963T).

Distribution, sources and ecological risk assessment of PAHs in historically contaminated surface sediments at Bhavnagar coast, Gujarat, India.

The concentration, distribution and ecological risk of polycyclic aromatic hydrocarbons (PAHs) have been investigated in surface sediments near Bhavnagar coast. The concentration of ∑PAHs ranged from 5.02 to 981.18 µg g(-1) dry weight, indicating heavy pollution compared to other historically polluted study sites. It was found to be introduced via mixed origins such as burning of gas, oil, coal, production of petrochemicals, cement, and rubber tires. Domestic fuel burning and motor vehicles are also culprits for air pollution. Industrial effluents and accidental oil spillage can also be considered. PAHs can be exposed through air, water, soil and food sources including ingestion, inhalation, and dermal content in both occupational and non-occupational levels by single or sometimes multiple exposures routes concomitantly. Furthermore, diagnostic ratios, statistical principal component analysis (PCA) and hierarchical cluster analysis (HCA) models have confirmed that the sources of PAHs were both - petrogenic and pyrogenic. For both the sites, assessment of ecological risk of the elevated levels of these pollutants has been exercised based on toxic equivalency factors (TEFs) and risk quotient (RQ) methods. The composite results indicated accurately that both the sites, bears potentially acute and chronic health hazards such as decreased immune functionality, genotoxicity, malignancy and developmental malfunctions in humans. The sites studied here and the workers have been exposed to hazardous pollutants for a longer period of time. Evidences indicate that mixtures of PAHs are carcinogenic to humans, based on occupational studies on workers, exposed to these pollutants. Hence, the present study and statistical approaches applied herein clearly indicate the historic mix routes of PAHs that resulted in magnified concentrations leading to high ecosystem risk. Thus, the scientific communities are urged to develop strategies to minimize the concentrations of PAHs from the historically impacted coastlines, thereby concerning for the future investigations and restoration of these sites.

Enhanced chrysene degradation by a mixed culture Biorem-CGBD using response surface design.

Degradation of chrysene, a four ringed highly carcinogenic polycyclic aromatic hydrocarbon (PAH) has been demonstrated by bacterial mixed culture Biorem-CGBD comprising Achromobacter xylosoxidans, Pseudomonas sp. and Sphingomonas sp., isolated from crude oil polluted saline sites at Bhavnagar coast, Gujarat, India. A full factorial Central Composite Design (CCD) using Response Surface Methodology (RSM) was applied to construct response surfaces, predicting 41.93% of maximum chrysene degradation with an experimental validation of 66.45% chrysene degradation on 15th day, using a combination of 0.175, 0.175 and 0.385 mL of OD600 = 1 inoculum of A. xylosoxidans, Pseudomonas sp. and Sphingomonas sp., respectively and a regression coefficient (R2) of 0.9485 indicating reproducibility of the experiment. It was observed that chrysene degradation can be successfully enhanced using RSM, making mixed culture Biorem-CGBD a potential bioremediation target for PAH contaminated saline sites.

Bacillus crescens sp. nov., isolated from soil.

Two bacterial strains (JC247T and JC248) were isolated from soil samples collected from Rann of Kutch, Gujarat, India. Colonies of both strains were creamy white. Cells were Gram-stain-positive, rods-to-curved rods (crescent-shaped), and produced centrally located oval-shaped endospores. Major (>5 %) fatty acids of both strains were iso-C16  :  0, iso-C14  :  0, iso-C15  :  0, C16  :  1ω11c and C16  :  0, with minor ( < 5 but >1 %) amounts of anteiso-C15  :  0, anteiso-C17  :  0, iso-C16  :  1 H, iso-C17  :  0, iso-C18  :  0, C14  :  0, C17  :  0, C18  :  0, C18  :  1ω9c, iso-C17  :  1ω10c and anteiso-C17  :  0B/isoI. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids of both strains. Cell-wall amino acids were l-alanine, d-alanine, d-glutamic acid and meso-diaminopimelic acid. The genomic DNA G+C content of strains JC247T and JC248 was 48.2 and 48.1 mol%, respectively. Both strains were closely related with mean DNA-DNA hybridization >90 %. 16S rRNA gene sequence analysis of both strains indicated that they are members of the genus Bacillus within the family Bacillaceae of the phylum Firmicutes. Both strains had a 16S rRNA gene sequence similarity of 96.93 % with Bacillus firmus NCIMB 9366T and < 96.92 % with other members of the genus Bacillus. Sequence similarity between strain JC247T and JC248 was 100 %. Distinct morphological, physiological and genotypic differences from previously described taxa support the classification of strains JC247T and JC248 as representatives of a novel species of the genus Bacillus, for which the name Bacilluscrescens sp. nov. is proposed. The type strain is JC247T ( = KCTC 33627T = LMG 28608T).

Polyhydroxyalkanoate from marine Bacillus megaterium using CSMCRI's Dry Sea Mix as a novel growth medium.

Oceans have significant potential to empower mankind and thus marine organisms are believed to be an enormous source for useful biomolecules. Polyhydroxyalkanoates (PHAs) are biological macromolecules that can be applied in nearly all fields. In the present study, Bacillus megaterium strain JK4h has been exploited for maximum PHB production using novel Dry Sea Mix (DSM) via Central Composite Design (CCD) of Response Surface Methodology (RSM) approach. The isolate was found to be producing 56.77% Cell Dry Weight (CDW) of PHAs within 24h, with optimized combinations of peptone, yeast extract and glucose. The PHB yield had been increased 2.61 fold compared to un-optimized experiments. The obtained PHA/PHB had been chemically characterized through Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR), Thermo Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). The results indicate the successful optimization for maximum production of biological macromolecule and it was found to be highly pure polyhydroxybutyrate (PHB). Thus, DSM can be served as a novel and cost effective medium for PHA production offering the use of marine resources as a "green" sustainable alternative.

Application of response surface methodology for rapid chrysene biodegradation by newly isolated marine-derived fungus Cochliobolus lunatus strain CHR4D.

For the first time, Cochliobolus lunatus strain CHR4D, a marine-derived ascomycete fungus isolated from historically contaminated crude oil polluted shoreline of Alang-Sosiya ship-breaking yard, at Bhavnagar coast, Gujarat has been reported showing the rapid and enhanced biodegradation of chrysene, a four ringed high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH). Mineral Salt Broth (MSB) components such as ammonium tartrate and glucose along with chrysene, pH and trace metal solution have been successfully optimized by Response Surface Methodology (RSM) using central composite design (CCD). A validated, two-step optimization protocol has yielded a substantial 93.10% chrysene degradation on the 4(th) day, against unoptimized 56.37% degradation on the 14(th) day. The results depict 1.65 fold increase in chrysene degradation and 1.40 fold increase in biomass with a considerable decrement in time. Based on the successful laboratory experiments, C. lunatus strain CHR4D can thus be predicted as a potential candidate for mycoremediation of HMW PAHs impacted environments.